1 2 3 4 5

Materials and Methods
Lytchinus pitus or Strongylocentrotus purpuratus (from the Pacific coast), and Arbacia punctulata or Lytchinus variegates (from the Atlantic and Gulf of Mexico) can be used for this experiment. Lytchinus variegatus was used for our experiment. Sea urchin gamete release was induced by injection of KCl and eggs and sperm were obtained in separate beakers as described in basic protocol "sea urchin gamete collection".

Eggs were obtained in sulfate free (SF) water , to ensure that they did not incorporate sulfate. All eggs were then washed four times in SF water. Eggs were fertilized as described in the basic protocol "sea urchin fertilization". Eggs were checked for fertilization under the microscope. A beaker was filled with artificial sea water (ASW), and another beaker was filled with sulfate-free water. Fertilized eggs were then pipetted into each beaker. Embryos were incubated at about 24° C (room temperature) overnight. Since embryos were not fully developed by the next day, they were incubated for an additional day. On day two, because yield of hatched blastulas was low and embryos were dilute, swimming embryos floating at the top of the water were centrifuged and concentrated. These concentrated embryos were then fixed as described in the "Preparation of fixed embryos for immunocytochemistry and AP staining". Embryos were then divided into two groups and stained with 5C7, a monoclonal antibody that stains endoderm, as described in the "Immunofluorescent staining of Sea Urchin embryos" or histochemically stained as described in the "Histochemical staining of sea urchin embryos for alkaline phosphatase (AP) enzyme activity".

© 2001 Cebra-Thomas
Last Modified: 25 May 2004

[Lab Protocols | Students | Cebra-Thomas | Course | Links ]